P38 MAPK在滑膜细胞表达炎性因子中的作用
|
摘要
目的:研究p38丝裂原活化蛋白激酶(mitogen activated protein kinase,MAPK)在脂多糖(LPS)诱导大鼠滑膜成纤维细胞(synovial fibroblast,SF)合成炎性因子IL-1β、MMP3中的作用。方法:采用酶消化法分离获得大鼠SF,体外培养并鉴定,分别作为空白对照组、与不同浓度LPS共培养、以SB203580预处理后与LPS共培养,24h后免疫化学染色、RT-PCR分别检测通路活化水平和炎性因子蛋白及m RNA表达量。结果:LPS可刺激SF使IL-1β、MMP3蛋白和m RNA表达上调,表达量随LPS浓度增高而增加(与对照组相比P<0.05);且与LPS共培养后SF中p38 MAPK通路活化,阻断p38 MAPK后,炎性因子表达显著下调(P<0.05)。结论:LPS诱导SF高表达IL-1β、MMP3,p38 MAPK在其中发挥重要作用。
Objective: To investigate the effect of p38 MAPK on IL-1β and MMP3 expression in rat temporomandibular joint synovial fibroblasts induced by lipopolysaccharide. Method: Synovial fibroblasts were separated from rat TMJ by enzyme digestion then subcultured and the phenotypes were analyzed with cell surface markers vimentin and CD68. SF were respectively treated with different concentration of LPS for 24 hours or pretreated with SB203580 before LPS. The ex pressions of IL-1, MMP3, p-p38, p-ATF2 were detected by immunochemical staining, expressions of IL-1 and MMP3 m RNA were detected by RT-PCR to evaluated the activation level of p38 signal and gene and protein expression levels of inflammatory factor. The non-treated cells were used as control. Result: Compared with the control, the protein and m RNA expressions of IL-1 and MMP3 in SF treated with LPS were up-regulated and expression levels increased with the concentration of LPS(P<0.05). Activation level of p38 MAPK signal increased in the SF treated with LPS. The expressions of IL-1and MMP3 in the SF pretreated with p38 inhibitor SB203580 were significantly down-regulated P<0.05). Conclusion: LPS enhanced the expression of IL-1 and MMP3 in the SF in a dose-dependent manner, and p38 signal probably involved in the process.
Objective: To investigate the effect of p38 MAPK on IL-1β and MMP3 expression in rat temporomandibular joint synovial fibroblasts induced by lipopolysaccharide. Method: Synovial fibroblasts were separated from rat TMJ by enzyme digestion then subcultured and the phenotypes were analyzed with cell surface markers vimentin and CD68. SF were respectively treated with different concentration of LPS for 24 hours or pretreated with SB203580 before LPS. The ex pressions of IL-1, MMP3, p-p38, p-ATF2 were detected by immunochemical staining, expressions of IL-1 and MMP3 m RNA were detected by RT-PCR to evaluated the activation level of p38 signal and gene and protein expression levels of inflammatory factor. The non-treated cells were used as control. Result: Compared with the control, the protein and m RNA expressions of IL-1 and MMP3 in SF treated with LPS were up-regulated and expression levels increased with the concentration of LPS(P<0.05). Activation level of p38 MAPK signal increased in the SF treated with LPS. The expressions of IL-1and MMP3 in the SF pretreated with p38 inhibitor SB203580 were significantly down-regulated P<0.05). Conclusion: LPS enhanced the expression of IL-1 and MMP3 in the SF in a dose-dependent manner, and p38 signal probably involved in the process.
引文
[1] Schett G,Zwerina J,Firestein G.The p38 mitogen-activated protein kinase (MAPK) pathway in rheumatoid arthritis [J].Ann Rheum Dis,2008,67(7): 909-916
[2] Boileau C,Martel-Pelletier J,Brunet J,et al.PD-0200347,an α2δ ligand of the voltage gated calcium channel,inhibits in vivo activation of the Erk1/ 2 pathway in osteoarthritic chondrocytes: a PKCα dependent effect[J].Ann Rheum Dis,2006,65(5): 573-580
[3] Fan Z,Soder S,Oehler S,et al.Activation of interleukin-1 signaling cascades in normal and osteoarthritic articular cartilage[J].Am J Pathol,2007,171(3): 938-946
[4] 刘奉琴.低氧联合p38 MAPK 抑制剂SB203580 对LPS 诱导小鼠巨噬细胞TNF-α 表达的影响研究[D].山东大学,2008
[5] 郑培惠,赵华强,郑家伟.正常颞颌关节滑膜的组织学研究[J].上海口腔医学,1998,7(1): 54-56
[6] Wilkinson L S,Pitsillides A A,Worrall J G,et al.Light microscopic characterization of the fibroblast-like synovial intimal cell (synoviocyte) [J].Arthritis Rheum,1992,35 (10):1179-1184
[7] Sellam J,Berenbaum F.The role of synovitis in pathophysiology and clinical symptoms of osteoarthritis [J].Nat Rev Rheumatol,2010,6(11): 625-635
[8] Wang X D,Kou X X,Mao J J,et al.Sustained inflammation induces degeneration of the temporomandibular joint [J].JDent Res,2012,91(5): 499-505
[9] Mor A,Abramson S B,Pillinger M H.The fibroblast-like synovial cell in rheumatoid arthritis: a key player in inflammation and joint destruction[J].Clin Immunol,2005,115(2):118-128
[10] 徐婧秋,吴丽萍.脂多糖和白细胞介素-1β 对人牙周膜细胞中诱导型一氧化氮合酶表达的影响[J].华西口腔医学杂志,2011,29(4): 415-419
[11] Kaneyama K,Segami N,Sun W,et al.Analysis of tumor necrosis factor-α,interleukin-6,interleukin-1β,soluble tumor necrosis factor receptors I and II,interleukin-6 soluble receptor,interleukin-1 soluble receptor type II,interleukin-1 receptor antagonist,and protein in the synovial fluid of patients with temporomandibular joint disorders[J].Oral Surg Oral Med Oral Pathol Oral Radiol Endod,2005,99(3): 276-284
[12] Xiu Z B,Chen W T,Sun K M.Study on correlation between the pathological changes under arthroscopy and the cytokine levels in the knee osteoarthritis of the Blood Stasis type [J].Zhongguo Gu Shang,2010,23(12): 890-893
[13] 吴庆亭,孔静静,杨盈盈,等.四种建立颞下颌关节滑膜炎动物模型方法的比较[J].口腔颌面修复学杂志,2014,15(4):210-213
[15] Zarubin T,Jiahuai H A N.Activation and signaling of the p38 MAP kinase pathway[J].Cell Res,2005,15(1): 11-18
[16] 于波,朱振安.骨关节炎治疗靶点p38MAPK 信号通路研究进展[J].国际骨科学杂志,2011,32(6): 343-345
[17] Lisnock J M,Tebben A,Frantz B,et al.Molecular basis for p38 protein kinase inhibitor specificity [J].Biochemistry,1998,37(47): 16573-16581
[2] Boileau C,Martel-Pelletier J,Brunet J,et al.PD-0200347,an α2δ ligand of the voltage gated calcium channel,inhibits in vivo activation of the Erk1/ 2 pathway in osteoarthritic chondrocytes: a PKCα dependent effect[J].Ann Rheum Dis,2006,65(5): 573-580
[3] Fan Z,Soder S,Oehler S,et al.Activation of interleukin-1 signaling cascades in normal and osteoarthritic articular cartilage[J].Am J Pathol,2007,171(3): 938-946
[4] 刘奉琴.低氧联合p38 MAPK 抑制剂SB203580 对LPS 诱导小鼠巨噬细胞TNF-α 表达的影响研究[D].山东大学,2008
[5] 郑培惠,赵华强,郑家伟.正常颞颌关节滑膜的组织学研究[J].上海口腔医学,1998,7(1): 54-56
[6] Wilkinson L S,Pitsillides A A,Worrall J G,et al.Light microscopic characterization of the fibroblast-like synovial intimal cell (synoviocyte) [J].Arthritis Rheum,1992,35 (10):1179-1184
[7] Sellam J,Berenbaum F.The role of synovitis in pathophysiology and clinical symptoms of osteoarthritis [J].Nat Rev Rheumatol,2010,6(11): 625-635
[8] Wang X D,Kou X X,Mao J J,et al.Sustained inflammation induces degeneration of the temporomandibular joint [J].JDent Res,2012,91(5): 499-505
[9] Mor A,Abramson S B,Pillinger M H.The fibroblast-like synovial cell in rheumatoid arthritis: a key player in inflammation and joint destruction[J].Clin Immunol,2005,115(2):118-128
[10] 徐婧秋,吴丽萍.脂多糖和白细胞介素-1β 对人牙周膜细胞中诱导型一氧化氮合酶表达的影响[J].华西口腔医学杂志,2011,29(4): 415-419
[11] Kaneyama K,Segami N,Sun W,et al.Analysis of tumor necrosis factor-α,interleukin-6,interleukin-1β,soluble tumor necrosis factor receptors I and II,interleukin-6 soluble receptor,interleukin-1 soluble receptor type II,interleukin-1 receptor antagonist,and protein in the synovial fluid of patients with temporomandibular joint disorders[J].Oral Surg Oral Med Oral Pathol Oral Radiol Endod,2005,99(3): 276-284
[12] Xiu Z B,Chen W T,Sun K M.Study on correlation between the pathological changes under arthroscopy and the cytokine levels in the knee osteoarthritis of the Blood Stasis type [J].Zhongguo Gu Shang,2010,23(12): 890-893
[13] 吴庆亭,孔静静,杨盈盈,等.四种建立颞下颌关节滑膜炎动物模型方法的比较[J].口腔颌面修复学杂志,2014,15(4):210-213
[15] Zarubin T,Jiahuai H A N.Activation and signaling of the p38 MAP kinase pathway[J].Cell Res,2005,15(1): 11-18
[16] 于波,朱振安.骨关节炎治疗靶点p38MAPK 信号通路研究进展[J].国际骨科学杂志,2011,32(6): 343-345
[17] Lisnock J M,Tebben A,Frantz B,et al.Molecular basis for p38 protein kinase inhibitor specificity [J].Biochemistry,1998,37(47): 16573-16581